INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY WORLD HEALTH ORGANIZATION Toxicological evaluation of certain veterinary drug residues in food WHO FOOD ADDITIVES SERIES 39 Prepared by: The forty-eighth meeting of the Joint FAO/WHO Expert Committee on Food Additives (JECFA) World Health Organization, Geneva 1997 DANOFLOXACIN First draft prepared by Dr E.A.M. Good Veterinary Medicines Directorate Addlestone, United Kingdom 1. Explanation 2. Biological data 2.1 Biochemical aspects 2.1.1 Absorption, distribution, and excretion 2.1.2 Biotransformation 2.2 Toxicological studies 2.2.1 Acute toxicity 2.2.2 Short-term toxicity 2.2.3 Long-term toxicity and carcinogenicity 2.2.4 Genotoxicity 2.2.5 Reproductive toxicity 2.2.5.1 Multigeneration reproductive toxicity 2.2.5.2 Developmental toxicity 2.2.6 Special study on delayed contact hypersensitization 2.2.7 Special studies on pharmacological activity 2.2.8 Special studies on microbiological effects 2.3 Observations in humans 3. Comments 4. Evaluation 5. References 1. EXPLANATION Danofloxacin is a 'third-generation' fluoroquinolone antibiotic. It is manufactured by a stereospecific synthesis, resulting in the pure S (laevorotatory) form. Danofloxacin is used in veterinary medicine as the mesylate salt for the treatment of respiratory diseases in cattle, swine, and chickens. All of the studies of toxicity were carried out with the mesylate salt of dano-floxacin. The doses are expressed in terms of the active moiety, the zwitterion base. Desmethyldanofloxacin, which is the primary metabolite of danofloxacin, has similar properties. The toxicity and antimicrobial activity of desmethyl-danofloxacin were also examined, again using the mesylate salt. The structures of danofloxacin and desmethyldanofloxacin are shown in Figure 1. Danofloxacin has not previously been reviewed by the Joint FAO/WHO Expert Committee on Food Additives.2. BIOLOGICAL DATA 2.1 Biochemical aspects 2.1.1 Absorption, distribution, and excretion Chickens Groups of male 18-day-old broiler chickens were given drinking-water that had been medicated to provide 5 mg/kg bw per day danofloxacin, for three days. Steady-state concentrations of 0.43 and 0.21 µg/ml were obtained in lung and plasma, respectively, during the period of administration. The half-life of danofloxacin was 4.9 h in plasma and 5.8 h in lung. No residues of desmethyldanofloxacin were detected in any of the plasma or lung samples (Lynch et al., 1990a). Groups of broiler chickens were given drinking-water that had been medicated to provide 5 mg/kg bw per day 3H-danofloxacin for five days. The test substance was tritiated in the N1-cyclopropyl ring. The study complied with GLP guidelines. Six hours after withdrawal of the treated water, unchanged danofloxacin accounted for 75% of the radiolabel in excreta, and desmethyl-danofloxacin accounted for 5-7% (Lynch et al., 1990a). Groups of 12 male and 12 female 22-day-old broiler chickens were given drinking-water which had been medicated to provide 5 mg/kg bw per day 3H-danofloxacin, for five days. The test substance was tritiated in the N1-cyclo-propyl ring and had a specific activity of 12.1 µCi/mg (expressed as base). Excreta were collected every 24 h and pooled by sex and day. The study complied with GLP guidelines. The mean level of radiolabel in excreta attained a steady state in the range 35-46 µg/g by the first day of treatment but declined to 20 and 6.6 µg/g on the first and second days after withdrawal, respectively. About 85% of the radiolabel in faeces was identified as unmetabolized danofloxacin. There was no difference between the sexes in the pattern of excretion (Lynch et al., 1988). Pigs Castrated male and female pigs (mean body weight, 40 kg) were given single intramuscular injections of 1.25 mg/kg bw of a prototype 2.5% formulation of danofloxacin. Absorption was rapid, with peak concentrations of 0.40 µg/g in plasma and 1.68 µg/g in lung about 1 h after dosing. The half-life of plasma elimination was approximately 7 h (Risk et al., 1991). Six castrated male and six female pigs (mean body weight, 43.3 kg) were given five daily intramuscular injections of 1.25 mg/kg bw of danofloxacin tritiated in the N1-cyclopropyl ring in a 2.5% aqueous vehicle. The study complied with GLP guidelines. The mean radiolabel concentration during the two days before and the day after the last of the five doses was 7-8 µg/g in pooled faecal homogenates and 14-18 µg/g in pooled urine samples. During this same period, the total daily recovery of radiolabel from urine and faeces accounted for 75-81% of the daily dose. The mean radiolabel concentration in bile was 1.7 µg/g 12 h after the end of treatment and had declined to 0.21 µg/g by 48 h (Lynch et al., 1989b). In pigs given a single dose of 5 mg/kg bw of danofloxacin intravenously, intramuscularly, or orally, detectable residues of danofloxacin appeared rapidly in plasma. A peak plasma concentration of 0.42 µg/ml was attained about 3 h after oral dosing. The bioavailability was estimated to be 76% after intramuscular administration and 89% after oral administration (Mann & Frame, 1992). Cattle A group of 36 male and female calves weighing about 250 kg were given a single intramuscular injection of 1.25 mg/kg bw of a 2.5% prototype formulation of danofloxacin, and the kinetics of the compound were evaluated in plasma and lung tissues. The study complied with GLP guidelines. Absorption was rapid, with peak plasma and lung concentrations of 0.35 µg/ml and 1.44 µg/g attained 1 h after dosing. The half-time for plasma elimination was 3.4 h (Lynch et al., 1990b). In a three-way cross-over design study, 12 male and female beef calves (mean body weight, 112 kg) were given single intravenous doses or five subcutaneous or intramuscular doses of 1.25 mg/kg bw per day of a 2.5% prototype formulation of danofloxacin. The study complied with GLP guidelines. Absorption was rapid: peak plasma concentrations of 0.37 and 0.47 µg/ml were attained about 1 h after single subcutaneous and intramuscular doses, respectively, and the bioavailability was almost 100%. The values for the area under the concentration-time curve indicated that the levels achieved after intramuscular and subcutaneous administration were bioequivalent after one, three, and five injections (Lynch et al., 1991a). The pattern of excretion of 3H-danofloxacin (tritiated in the N1-cyclopropyl ring) was studied in five castrated male and four female calves (body weight, about 180 kg) after five daily intramuscular injections of 1.25 mg/kg bw (in a 2.5% aqueous vehicle). The study complied with GLP guidelines. The total concentration of danofloxacin-related material in excreta reached a plateau by the third day of treatment. Approximately equal amounts were excreted in urine and faeces. Unchanged danofloxacin accounted for about 48% of material excreted in faeces and 89% in urine. The desmethyl metabolite accounted for 12% of the material in urine samples, but the concentrations in faeces were too low for measurement (Lynch et al., 1989a). 2.1.2 Biotransformation The biotransformation of danofloxacin has been investigated in rats, dogs, chickens, pigs, and cattle. In all of these species, the main residue in the faeces was unmetabolized danofloxacin; smaller amounts of desmethyldanofloxacin were also found (Lynch et al., 1989a,b,c, 1991a,b,c,d). The main residue in the urine of cattle, dogs, and rats was also danofloxacin. Desmethyldanofloxacin, danofloxacin- N-oxide and the ß-glucuronide were identified in rat, dog, and pig urine (Lynch et al., 1991d). In the livers of most species, danofloxacin was the major residue, but significant residues of the metabolite desmethyldanofloxacin were also present (Table 1). A piperazine-ring degradation product constituted 43 and 27% of the radiolabel in bile from male and female cattle, respectively, but it was present in only trace amounts in bile from rats and dogs. The bile of dogs contained residues of danofloxacin- N-oxide, which was present in only trace amounts in bile from the other species (Lynch et al., 1991d). 2.2 Toxicological data 2.2.1 Acute toxicity The results of studies of the acute toxicity of danofloxacin and desmethyl-danofloxacin are summarized in Table 2. All of the studies complied with GLP guidelines. In the studies by oral administration, signs of toxicity included exophthalmia, decreased activity, tremors, and twitching, which occurred within a few minutes of dosing. One of three mice given 2000 mg/kg bw died 29 min after dosing. Retching, clonic convulsions, and gasping were observed before death. After intravenous dosing, all three mice given 100 mg/kg bw and all three rats given 150 mg/kg bw died within 3 min. The signs of toxicity were similar to those produced after oral administration. Similar signs of toxicity were observed after oral and intravenous dosing with desmethyldanofloxacin. Table 1. Composition of residues in liver in five species given danofloxacin Species Dose regimen Sex Total Danofloxacina Desmethyldanofloxacina Reference residue Rat 5 × 6.25 mg/kg bw M 2.3 ppm 67% 18% Lynch et al. per day orally F 2.5 ppm 70% 11% (1991d) Dog 5 × 2.4 mg/kg bw M 6.7 ppm 66% 28% Lynch et al. per day orally F 5.4 ppm 58% 37% (1991d) Chicken 5 × 5 mg/kg bw M NR 78% 16% Lynch et al. per day orally F NR 74% 12% (1988) Pig M 0.95 ppm 58% 23% Lynch et al. F 0.96 ppm 68% 29% (1989c) Cowb 5 × 1.25 mg/kg bw M 0.82 ppm 24% 43% Lynch et al. intramuscularly F 1.1 ppm 29% 42% (1991d) a Relative to the radiolabel recovered by high-performance liquid chromatography b Killed 12 h after the last dose Table 2. Results of studies of the acute toxicity of danofloxacin and desmethyldanofloxacin Species Route Vehicle Sex LD50 Reference (strain) (mg/kg bw) Danofloxacin Rat (Sprague- Oral Distilled M & F > 2000 Stadnicki et al. (1988a) Dawley) water Mouse (ICR) Oral Distilled water M & F > 2000 Stadnicki et al. (1988a) Rat (Sprague- Intravenous Sterile water M 100-150 Stadnicki et al. (1988a) Dawley) Mouse (ICR) Intravenous Sterile water M 50-100 Stadnicki et al. (1988a) Rabbit (New Dermal M & F > 299a Stadnicki et al. (1988b) Zealand white) Desmethyldanofloxacin Rat (Sprague- Oral Deionized M & F > 2000 Stadnicki et al. (1989) Dawley) water Mouse (ICR) Oral Deionized M > 2000 Stadnicki et al. (1989) water Mouse (ICR) Oral Deionized F 1500-2000 Stadnicki et al. (1989) water Rat (Sprague- Intravenous Sterile water M 40-50 Stadnicki et al. (1989) Dawley) Mouse (ICR) Intravenous Sterile water M 7.5-10 Stadnicki et al. (1989) a 0.5 g danofloxacin was applied to one site on intact skin and one on abraded skin of two male and one female New Zealand white rabbits under an occlusive dressing for 24 h. There were no deaths or signs of toxicity, except for mild erythema at all treated sites. A dose of 26 mg danofloxacin (in a volume of 0.1 ml) was instilled into the conjunctival sac of the left eyes of three New Zealand white rabbits. The eyes were not rinsed after dosing. Mild conjunctivitis and a colourless discharge were observed within 1 h of treatment. All signs had cleared within 96 h (Stadnicki et al., 1988b). 2.2.2 Short-term toxicity Mice In a study designed to identify suitable doses for a two-year feeding study, groups of 15 male and 15 female Crl:CD-1 (ICR)BR VAF/Plus mice were fed diets calculated to provide 0, 150, 300, or 600 mg/kg bw per day of danofloxacin (74% activity) for up to 102 days. The study complied with GLP guidelines. One female given the high dose was killed in a moribund condition. Body-weight gain was reduced in males at the low and high doses in comparison with that of the controls. Decreased erythrocytic parameters were seen occasionally in males and females at the high dose. There were no consistent dose-related changes in clinical chemistry. At termination, the absolute mean group weight of the kidneys of females receiving the high dose was significantly increased, but there were no corresponding pathological changes. Microscopic examination revealed caecal dilatation in six mice at the low dose, 23 at the intermediate dose, and 27 at the high dose, but not in the controls. Inflammation of the caecum was seen in two mice at the high dose, and Gram-positive bacteria, tentatively identified as Clostridium difficile, were found in the inflamed caeca of both animals. Thymic lymphocytosis was seen in one of 15 females at the low dose, two of 15 at the intermediate dose, and eight of 15 at the high dose and in three of 13 males at the intermediate dose and six of 14 at the high dose but in no control. A NOEL was not identified (Stadnicki et al., 1994a). Rats A two-week exploratory study was carried out in which groups of three male and three female Long-Evans rats received oral doses of 0, 25, 50, or 100 mg/kg bw per day of danofloxacin in an aqueous vehicle. There were no significant effects on body-weight gain, food consumption, haematological, clinical chemical, or urinary parameters, or organ weights. At necropsy, treatment-related caecal enlargement was observed in treated animals (Reynolds et al., 1987a). Groups of 10 male and 10 female Long-Evans rats were given doses of 0, 25, 75, or 150 mg/kg bw per day danofloxacin by gavage in an aqueous vehicle for one month. Serum chemistry and haematology were evaluated before treatment and on days 11-12 and 30-31. The study complied with GLP guidelines. There were no signs of toxicity and no effects on body-weight gain or food consumption. Alanine aminotrasferase levels were significantly increased in treated females receiving the high dose, and absolute liver and liver:body weight ratios were significantly decreased in males at this dose. No substance-related effects were seen in about 30 tissues from animals at 0 and 150 mg/kg bw and macroscopic lesions from animals in the other groups examined microscopically (Fisher et al., 1988a). In a range-finding study designed to identify suitable doses for a long-term study of toxicity, groups of 10 male and 10 female Fischer rats were fed diets calculated to provide 0, 150, 300, 450, or 600 mg/kg bw per day of danofloxacin for 36-39 days. Satellite groups of three animals of each sex were used to monitor plasma concentrations of the drug. Five rats given 600 mg/kg bw per day died. Body-weight gain and food consumption were reduced in all treated groups, and there was an initial dose-related decrease in food consumption. Changes were seen in serum chemistry, haematological parameters and organ weights, due to the effects of starvation and dehydration. Caecal dilatation was observed in all treated animals (Stadnicki et al., 1994b). In a subsequent exploratory study in groups of 10 Long-Evans rats of each sex, doses intended to provide 0, 75, 150, or 300 mg/kg bw per day of danofloxacin were administered in the feed for seven days; the animals then received untreated feed for seven days and the treated feed for a further 12 days. Body-weight gain and food consumption were reduced in rats at the intermediate and high doses. Serum globulin levels were reduced and leukopenia was observed in all treated groups. Crystalluria was seen in most treated rats but in only two of 20 controls. There was no histopathological evidence of nephropathy (Stadnicki et al., 1994c). In a study designed to establish doses to be used in a two-year feeding study, groups of 15 Long-Evans rats of each sex were fed diets calculated to provide 0, 75, 150, or 300 mg/kg bw per day of danofloxacin for about three months. The study complied with GLP guidelines. Alopecia was seen in four females at the high dose and one at the intermediate dose. Discoloured urine (especially in animals at 75 mg/kg bw) and crystalluria were observed in all treated groups, but there were no corresponding pathological changes in the kidneys. Body-weight gain was significantly reduced in a dose-related manner. There were significant, dose-related increases in urinary and serum magnesium concentrations, which were attributed to chelation of magnesium ions by the test substance. Caecal dilatation was observed in rats at 150 and 300 mg/kg bw. A range of degenerative changes were observed in the testes of all males at the high dose and in one at 0, one at 75, and one at 150 mg/kg bw per day. Degenerating germinal cells were found in the epididymides of all males at the high dose and nine at the middle dose (Stadnicki et al., 1994d). Groups of 20 male and 20 female weanling Long-Evans rats were selected from the F1 offspring of rats used in a multigeneration study which had been exposed to danofloxacin both in utero and during lactation. They were then given oral doses of 25, 75, or 150 mg/kg bw per day for up to three months, whereas 20 controls of each sex received deionized water. The study was conducted in accordance with US FDA GLP guidelines (21 CFR Part 58). There were no substance-related deaths and no adverse effects on food consumption or body-weight gain. There were no consistent dose-related trends in haematological or clinical chemical values. A dose-related increase in proteinuria was seen in females but not males, which was correlated with the finding of tubular nephropathy in individual animals. Kidney weights were unaffected by treatment. The mean absolute and relative weights of the testes of rats at 75 and 150 mg/kg bw were about 10% lower than those of controls. Lesions of the heart were found primarily in animals at these doses and consisted of either multifocal myocardial degeneration and necrosis, or multifocal fibrosis, or both. There was no NOEL, as renal tubular nephropathy was observed in all treated females (Fisher et al., 1989a). The study was repeated at lower doses in order to establish a NOEL. Groups of 20 male and 20 female weanling Long-Evans rats were again selected from the F1 offspring in the multigeneration study, which had been exposed to danofloxacin both in utero and during lactation. They were then given oral doses of 1, 2.5, or 6.25 mg/kg bw per day for up to three months. Twenty controls of each sex were given deionized water. The study was conducted in accordance with US FDA GLP guidelines (21 CFR Part 58). There were no treatment-related deaths or signs of toxicity. Body-weight gain, food consumption, and haematological and clinical chemical parameters were unaffected by treatment. Urinalysis revealed a marginal increase in the incidence of haematuria in males given the high dose and of proteinuria in a single female at this dose. The absence of any corresponding pathological changes in the kidney indicates that the findings reflected biological variability rather than a substance-related effect. The NOEL was 6.25 mg/kg bw per day (Fisher et al., 1990a). Groups of 20 male and 20 female Long-Evans rats were given oral doses of 1, 2.5, or 6.25 mg/kg bw per day of desmethyldanofloxacin for up to three months. The rats were selected from the F1 offspring in a multigeneration study and had been exposed to the test substance both in utero and during lactation. Groups of 20 controls of each sex received deionized water. The study was conducted in accordance with US FDA GLP guidelines (21 CFR Part 58). There were no effects on mortality, body-weight gain, food consumption, haematological or clinical chemical parameters, organ weights, or gross or histo-pathological findings. The NOEL was 6.25 mg/kg bw per day (Stadnicki et al., 1990a). Rabbits In an exploratory study, groups of three female New Zealand white rabbits were given oral doses of 0, 25, 50, or 100 mg/kg bw per day of danofloxacin in an aqueous vehicle. Owing to significantly reduced feed intake and decreased body weight, treatment of rabbits at the low dose was stopped after 12 doses, that of animals at the intermediate dose after seven doses, and that of rabbits at the high dose after four doses. The groups were fed untreated diet for a further five to eight days, then killed and necropsied. Pathological examination revealed a dose-related incidence of enlarged caeca (Lundeen et al., 1993). Dogs In an exploratory study, groups of one male and one female beagle dogs were given oral doses of 0, 12.5, 25, or 50 mg/kg bw per day of danofloxacin in gelatin capsules for two weeks. The female at the high dose had reduced body-weight gain and food consumption and increased serum blood urea nitrogen and creatinine levels. At necropsy, this dog had acute segmental nephrosis and renal failure. The male at the high dose had chronic focal necrosis without renal failure (Reynolds et al., 1987b). In a one-month exploratory study, groups of three male and three female beagle dogs (10-12 months old) were given oral doses of 0, 5, 10, or 25 mg/kg bw per day of danofloxacin in gelatin capsules. The study was conducted in accordance with US FDA GLP guidelines (21 CFR Part 58). There were no effects on behaviour, food consumption, body-weight gain, blood pressure, electrocardiographic profile, or ophthalmic, serum chemical, or haematological parameters. At necropsy, a full range of tissues from all animals was examined, with particular attention to the weight-bearing joints. Shallow erosions were found in all dogs, including controls, although the grade of erosions was slightly higher in those at the high dose. The lesions were typical of those found in older dogs and did not resemble quinolone-induced lesions (Fisher et al., 1988b). Groups of four beagle dogs of each sex aged about six months were given oral doses of 0, 5, 10, or 25 mg/kg bw of danofloxacin in gelatin capsules. The substance was administered as two equally divided doses per day for three months. At termination, all dogs were subjected to a complete pathological examination. The study was conducted in accordance with US FDA GLP guidelines (21 CFR Part 58). By day 7, reduced activity and signs of joint pain were seen in all eight dogs given 25 mg/kg bw and in three dogs given 10 mg/kg bw. Most of the affected animals showed signs of recovery by week 6, despite continued treatment. Body-weight gain, food consumption, heart rate, and respiratory rate were decreased in animals that showed clinical signs. There were no effects on electrocardiographic profiles, blood pressure, or ophthalmic, haematological, clinical chemical, or urinary parameters. Gross pathological changes were observed in the articular cartilage of the major joints of all treated animals, except for one female receiving the low dose. The lesions were characterized by areas of cartilage separation and cartilage loss (erosions). The severity of the lesions was dose-related. Microscopic examination revealed further changes, including the apparent alteration of collagen fibres when viewed with polarized light (Fisher et al., 1989b). A second three-month study, which complied with the same GLP guidelines, was carried out at lower doses in order to establish a NOEL. Groups of four male and four female beagle dogs, aged about five months, were given oral doses of 0, 1, or 2.4 mg/kg bw per day of danofloxacin in gelatin capsules as two equally divided doses, for 91 days. There were no signs of toxicity and no effects on body-weight gain, food consumption, clinical chemical or urinary parameters, or organ weights. There were no substance-related pathological findings. The NOEL was 2.4 mg/kg bw per day (Fisher et al., 1989c). Groups of three male and three female beagle dogs, four to six months old, were given oral doses of 0, 2.5, 5, or 10 mg/kg bw per day desmethyldanofloxacin in gelatin capsules, administered as two equally divided doses, for about three months The study was conducted in accordance with US FDA GLP guidelines (21 CFR Part 58). There were no notable effects on body-weight gain, electrocardio-graphic profiles, serum chemical or haematological values, or organ weights. One male given the high dose showed signs of pain on day 65 when pressure was applied to the left ankle joint. One female at the low dose showed signs of pain on day 92 when pressure was applied to the inside of the left upper hind limb. At termination, histopathological changes typical of quinolone-induced lesions were found in the articular cartilage of the right femoral condyle of one male given the high dose (Stadnicki et al., 1990b). Groups of three male and three female beagle dogs, five to six months old, were given oral doses of 0, 0.25 or 0.5 mg/kg bw per day of desmethyldanofloxacin in gelatin capsules, as two equally divided doses, for about three months. Further groups of dogs were given 10 mg/kg bw per day of danofloxacin. The study was conducted in accordance with US FDA GLP guidelines (21 CFR Part 58). There were no signs of toxicity in the dogs treated with desmethyldano-floxacin. The dogs given danofloxacin showed signs of hind limb weakness, reduced activity, and stiff gait. Treatment had no effect on electrocardiographic profiles, serum chemical or haematological parameters, or organ weights. Pathological examination revealed typical quinolone-induced arthropathy in all dogs treated with danofloxacin. One male in the group given 0.5 mg/kg bw per day desmethyldano-floxacin was found to have a single articular erosion in the patellar groove of the right knee. Microscopically, the erosion extended into zone 2 of the articular cartilage and resembled those seen with other quinolones. The NOEL was 0.25 mg/kg bw per day (Stadnicki et al., 1990c). 2.2.3 Long-term toxicity and carcinogenicity Mice Groups of 50 ICR mice of each sex were fed diets containing the equivalent of 10, 50, or 100 mg/kg bw per day of danofloxacin for up to two years. Two groups of 50 controls of each sex were fed untreated diets. The study complied with GLP guidelines. There were no adverse dose-related effects on mortality rate, body weight, food consumption, or haematological parameters. Females receiving 100 mg/kg bw per day gained significantly more weight than animals in the other groups. At termination, less than 50% of the animals in each group, except for the males receiving 10 mg/kg bw per day, were still alive. The mean absolute kidney weight (but not the relative kidney:body weight) was significantly increased in females receiving 100 mg/kg bw per day, which probably reflected the increased body weight of these mice. There was no evidence of carcinogenicity (Takatsu et al., 1995). Rats Groups of 50 male and 50 female Long-Evans rats were fed diets containing the equivalent of 10, 50, or 100 mg/kg bw per day of danofloxacin for up to two years. Two groups of 50 controls of each sex were fed untreated diets. The study complied with GLP guidelines. There were no clinical signs of toxicity, and survival was unaffected by treatment. At termination, however, less than 50% of animals in all groups were alive, except for females receiving 100 mg/kg bw per day and one of the groups of female controls. Occasional statistically significant reductions in body-weight gain were seen in males at this dose, and females at 50 and 100 mg/kg bw per day had significantly increased body-weight gain between three and 16 months of treatment, which was correlated with increased food intake during this period. Overall, the body-weight changes associated with treatment were minimal. Ophthalmoscopic examinations during weeks 12, 18, and 23 revealed no differences between control rats and those receiving 100 mg/kg bw per day. At termination, the mean leukocyte and neutrophil counts were significantly reduced in males receiving 100 mg/kg bw per day; in females at this dose, the mean haemoglobin, haematocrit, and lymphocyte counts were reduced. Males also had significant increases in aspartate aminotrans-ferase and sorbitol dehydrogenase values, and the mean globulin value was reduced, with a corresponding increase in albumin:globulin ratio. The sorbitol dehydrogenase level was also increased in males receiving 50 mg/kg bw per day. The effects on clinical chemical values were not seen in females. There were no substance-related effects on urinary values. The mean testis:body weight ratio was significantly reduced in the group receiving 100 mg/kg bw per day. There were no significant effects on absolute organ weights. Gross pathological examination revealed an increased incidence of caecal enlargement in treated animals which was not correlated with any microscopic findings. (Table 3). The histopathological findings included an increased incidence of renal papillary oedema, increased oligospermia, and abnormal content of the epididymides in rats at 100 mg/kg bw per day (Table 4). Table 3. Incidence of caecal enlargement in rats treated with danofloxacin in the diet for up to two years Sex Dose (mg/kg No. with caecal enlargement/ bw per day) no. examined Male 0 0/50 0 0/50 10 5/50 50 3/50 100 6/50 Female 0 1/50 0 0/50 10 1/50 50 1/50 100 2/50 Table 4. Incidence of some non-neoplastic histopathological alterations in groups of 50 rats fed diets containing danofloxacin for up to two years Sex Dose (mg/kg No. with papillary No. with No. with abnormal bw per day) oedema oligospermia epidydimal content Male 0 1 17 25 0 0 16 32 10 0 18 28 50 1 17 29 100 4 31 36 Female 0 0 0 2 10 2 50 2 100 4 There was a significant positive trend in the incidence of granular-cell tumours of the uterus and vagina in treated female rats (Table 5); however, the trend was not significant when corrected for multiple comparisons. Granular-cell foci were distinguished from tumours by their smaller size and the absence of compressed adjacent tissue; nevertheless, the morphology of these lesions was essentially the same. When the incidences of uterine and vaginal foci and tumours were combined, there was no statistically significant trend in the combined incidence across groups. There was a significant positive trend in the incidence of pituitary adenomas in female rats (Table 6), which was not significant when corrected for multiple comparisons. In addition, the incidence of pituitary adenomas was within the range seen in controls in the five previous studies at the same laboratory: 30/48 to 47/49 (Fisher et al., 1996). 2.2.4 Genotoxicity The results of assays for genotoxicity with danofloxacin and desmethyl-danofloxacin are summarized in Table 7. All of the studies complied with GLP guidelines. 2.2.5 Reproductive toxicity 2.2.5.1 Multigeneration reproductive toxicity Rats In a two-generation study of reproductive toxicity, groups of 45 male and 45 female Long-Evans rats were given oral doses of 0, 25, 75, or 150 mg/kg bw per day of danofloxacin in an aqueous vehicle. The study was conducted in accordance with US FDA GLP guidelines (21 CFR Part 58). Dams at 150 mg/kg bw per day showed reduced body-weight gain during gestation and fewer implantation sites and produced fewer live pups than those at other doses. The pup weights were significantly reduced at birth and during lactation. Similar effects were observed at the first F1 mating, which were more pronounced at the second F1 mating, when the pregnancy rate was adversely affected in all treated groups. The weights of all treated F2b pups were reduced in a dose-related manner (Fisher et al., 1990b). The F2b pups at 25 mg/kg bw per day were used in a new study (complying with similar GLP guidelines) when they were two months old, and treatment was continued. After mating to produce the F3 generation, the pregnancy rate was only 38%, whereas that in the control group was 65%. Post-implantation losses were significantly increased and pup body weights and survival were adversely affected (Fisher et al., 1990c). Table 5. Incidences of granular-cell proliferative lesions of the uterus and vagina in female rats fed diets containing danofloxacin for up to two years Lesion Dose (mg/kg bw per day) 0 0 10 50 100 Uterus No. examined 48 50 50 50 50 Granular-cell tumour 1 3 6 5 6 Granular-cell foci 5 6 1 1 2 Vagina 48 49 49 49 49 Granular-cell tumour 3 2 2 3 7 Granular-cemm foci 0 0 1 0 0 Uterus plus vagina Granular-cell tumour 4 5 8 8 13 Granular-cell foci5 5 6 2 1 2 Total 9 11 10 9 15 Table 6. Incidence of proliferative lesions of the pituitary gland in female rats fed diets containing danofloxacin for up to two years Lesion Dose (mg/kg bw per day) 0 0 10 50 100 No. examined 49 50 50 50 50 No. with pituitary adenoma 32 32 39 39 40 No. with pituitary hyperplasia 13 11 5 6 5 No. of rats alive at 24 months 22 26 19 18 26 Table 7. Assays for genotoxicity with danofloxacin and desmethyldanofloxacin End-point Test object Concentration S9 Results Reference Danofloxacin In vitro Gene mutationa S. typhimurium 0.01-0.2 µg/platea - Negative Amacher et al. TA98, TA100, 0.001-0.1 µg/plate + Negative (1988) TA1535, TA1537 0.0005-0.2 µg/plate + Negative Gene mutation L5178Y/tk+/- mouse 51-287 µg/ml - Negative Amacher et al. lymphoma cellsc 16-215 µg/ml + Negative (1988) Gene mutation hprt, Chinese 141-1070 µg/ml - Negative Guzzie et al. hamster ovary 465-2500 µg/ml + Negative (1992) cellsc Unscheduled Primary rat 50-400 µg/ml Negative Amacher et al. DNA synthesis hepatocytesc (1988) Cytogenetic Human lymphocytes 25-70 µg/ml - Positived Amacher et al. alterations 200-600 µg/ml + Positive (1990) In vivo Micronucleus ICR mouse bone 1000 mg/kg bw Negative Amacher et al. formation marrow orally (1990) Desmethyldanofloxacin In vitro Gene mutation S. typhimurium 0.001-0.5 µg/plate + Negative Amacher et al. TA98, TA100, 0.01-5 µg/plate - Negative (1991a) TA1535, TA1537 Gene mutation L5178Y mouse 90-388 µg/ml - Negative Holden et al. lymphoma cells 63-269 µg/ml +e Negative (1988) Unscheduled Primary rat 2.54-102 µg/ml and Positive Hazleton DNA synthesis hepatocytes 5.02-100 µg/ml Positive Laboratories America Inc. (1991a) Unscheduled Primary rat 62.5-250 µg/ml Positive Amacher et al. DNA synthesis hepatocytes 62.5-500 µg/mlf Positive (1991b) Table 7. (continued) End-point Test object Concentration S9 Results Reference In vivo Unscheduled Fischer 344 rat 1 × 250-2000 mg/kg Negative Hazleton DNA synthesis hepatocytes bw per day orally Laboratories America Inc. (1991b) Micronucleus CD-1 mouse bone 3 × 250-1000 Negative Amacher et al. formation marrow and peripheral mg/kg bw per day (1991c) blood orally S9, 9000 × g fraction of rat liver a The same bacterial strains were incubated with urine collected from mice given danofloxacin intraperitoneally at 5, 50, or 100 mg/kg bw per day, with no mutagenic response. b Higher concentrations toxic to tester strains c Not replicated independently d Significant increase in abnormal cells (chromatid breaks). To check whether the clastogenicity was due to chelation, the assay was repeated without S9 but with the addition of 400 µg/ml magnesium sulfate; no increase in abnormal cells was observed. In the presence of S9, extra washes to remove the test substance and the addition of magnesium sulfate diminished the clastogenic response. e From livers of uninduced rodents f 1.62 mmol/litre magnesium ions (as magnesium sulfate heptahydrate) added to culture medium did not prevent a similar dose-related increase in unscheduled DNA synthesis. In a three-generation study of reproductive toxicity, groups of 30 male and 30 female Long-Evans rats were given oral doses of 1, 2.5, 6.25, or 150 mg/kg bw per day of danofloxacin. Two control groups received the deionized water vehicle. Treatment was begun nine weeks before cohabitation for males and two weeks previously for females (1:1 mating) and was continued throughout gestation, parturition, and weaning of the F1 offspring. Of these, groups of 25 of each sex were selected randomly, and treatment was continued during breeding of the F2a and F2b generations. The F2b offspring were mated in the same way to produce the F3 litters. Litters were culled to eight pups on day 4 post partum. Postnatal development of the F2b pups was assessed at weaning by measuring locomotor activity, auditory function, and ophthalmic parameters. All F0, F1, and F2 rats used for breeding were necropsied and the reproductive organs and main target organs (kidney, joints, brain, heart, and liver) were weighed and preserved for possible microscopic examination. The study was conducted in accordance with US FDA GLP guidelines (21 CFR Part 58). Adult animals showed no treatment-related effects on survival, body-weight gain, food consumption, or clinical signs. In those at 150 mg/kg bw per day, copulatory rate and pregnancy rate were reduced and the length of gestation was increased. (The copulatory rate was defined as the percentage of those females which cohabited that had a vaginal flush with sperm, or an internal copulatory plug, or which delivered with no previous signs of mating.) Administration of 150 mg/kg bw per day also resulted in reduced litter size and pup weight at birth, reduced weight gain of the neonates, and a reduction in the number of pups surviving to day 4 post partum. The group receiving 150 mg/kg bw per day was terminated before the second F1 mating. The NOEL was 6.25 mg/kg bw per day (Stadnicki et al., 1990d). In a study of exactly the same design as that described above, the animals received desmethyldanofloxacin instead of danofloxacin. There were no treatment-related effects on parental body-weight gain or food consumption, clinical signs, pregnancy rate, length of gestation, number of implantation sites, or post-implantation loss. Pup body-weight gain and survival were unaffected by treatment. There were no grossly observed abnormalities in the dams or sires (Stadnicki et al., 1991). 2.2.5.2 Developmental toxicity Mice In a preliminary range-finding study, oral doses of 0, 50, 100, or 200 mg/kg bw per day of danofloxacin were given in an aqueous vehicle to groups of seven mated female ICR mice on days 6-13 of gestation. The study complied with GLP guidelines. The mean weights of the male fetuses of animals at 200 mg/kg bw per day were reduced. No effects were seen on dams (Kessedjian et al., 1988a). In the main study, groups of 20 female Crl:COBS-CDI(ICR)BR mice presumed to be pregnant were given oral doses of 0, 50, 100, or 200 mg/kg bw per day of danofloxacin in an aqueous vehicle. The dams were treated on days 6-13 of gestation and were killed on gestation day 18. A further group of 10 dams was given 200 mg/kg bw per day and used to monitor drug concentrations in maternal plasma and amniotic fluid. The study complied with GLP guidelines. The concentrations of danofloxacin in the amniotic fluid 5 h after the last of eight consecutive doses of 200 mg/kg bw per day were similar to those in maternal plasma, while those in fetal homogenates were two to three time those in maternal plasma. The incidence of non-gravid females was high: the total numbers of pregnant females were only 13, 16, 11, and 13 in the control, low, intermediate, and high dose groups, respectively. One dam given 200 mg/kg bw per day showed piloerection and prostration on days 7-10 of gestation and was found to have a cutaneous abscess at necropsy. The mean body-weight gain of the dams at the high dose was significantly reduced during treatment. There were no meaningful effects on the incidences of resorptions or fetal deaths or on the sex ratio. The mean weights of both male and female fetuses were significantly reduced at the high dose, and the incidence of delayed ossification was increased. There was no evidence of teratogenicity. The NOEL was 100 mg/kg bw per day, on the basis of maternal and fetal toxicity (Kesseddjian et al., 1989a). Rats In a preliminary range-finding study, oral doses of 0, 50, 100, or 200 mg/kg bw per day of danofloxacin were administered in an aqueous vehicle to groups of seven mated female Sprague-Dawley rats on days 6-15 of gestation. The study complied with GLP guidelines. Maternal body-weight gain was reduced in all treated groups, and the mean number of viable fetuses was reduced at 200 mg/kg bw per day (Kessedjian et al., 1988b). In the main study, groups of 20 female Sprague-Dawley rats presumed to be pregnant were given oral doses of 0, 50, 100, or 200 mg/kg bw per day of danofloxacin in an aqueous vehicle; 19-20 dams in each group were subsequently confirmed to be pregnant. The animals were treated on days 6-15 of gestation and were killed on gestation day 20. A further group of five dams was given 200 mg/kg bw per day and used to monitor drug concentrations in maternal plasma and amniotic fluid. The study complied with GLP guidelines. The concentrations of danofloxacin in the amniotic fluid 5 h after the last of eight consecutive doses of 200 mg/kg bw per day were similar to those in maternal plasma, while those in fetal homogenates were about three time those in maternal plasma. There were significant dose-related reductions in maternal body-weight gain in the rats at 100 and 200 mg/kg bw per day, and mean fetal weights were also significantly reduced. The incidences of delayed ossification and dilatation of the cerebral ventricles in the fetuses were significantly increased at the intermediate and high doses. The NOEL was 50 mg/kg bw per day, on the basis of maternal and fetal toxicity (Kesseddjian et al., 1989b). Rabbits In a preliminary range-finding study, groups of 11 New Zealand white rabbits presumed to be pregnant were given oral doses of 0, 5, 10, or 20 mg/kg bw per day of danofloxacin on days 6-20 of gestation. The study complied with GLP guidelines. Inappetance was seen in all groups, but maternal body weight was not affected. Animals at the high dose had a decreased number of live fetuses and higher resorption rates. The mean fetal weights were reduced at the intermediate and high doses (Tassinari et al., 1995a). In the main study, groups of 20 female New Zealand white rabbits presumed to be pregnant were given oral doses of 0, 2.5, 7.5, or 15 mg/kg bw per day of danofloxacin in an aqueous vehicle. The dams were treated on days 6-20 of gestation and were killed on gestation day 28. Because of a high incidence of non-gravid females, new animals were randomized to the different doses so that the total numbers were 32 controls, 29 at the low dose, 33 at the intermediate dose, and 39 at the high dose. The study complied with GLP guidelines. Eleven does at the high dose showed loss of body weight and reduced food consumption and then aborted their litters between days 22 and 28 of gestation. The mean food consumption of the does with viable litters at 15 mg/kg bw per day was significantly reduced during days gestation days 13-20. There were no treament-related effects on litter size, sex ratio, fetal weight, or the incidences of malformations or variations. The NOEL was 7.5 mg/kg bw per day, on the basis of maternal toxicity (Tassinari et al., 1995b). 2.2.6 Special study on delayed contact hypersensitization In a study using the Buehler closed patch technique, which complied with GLP guidelines, danofloxacin mesylate did not cause delayed contact hypersensitivity in the guinea-pig. A clear positive result was obtained with the known sensitizer, dinitrochlorobenzene (Beutler et al., 1992). 2.2.7 Special studies on pharmacological activity A number of pharmacological studies of danofloxacin were available that were not carried out in accordance with GLP guidelines or with any national or international guidelines. The main features of these studies are summarized in Table 8. Table 8. Results of pharmacological assays with danofloxacin Test system Doses Results Reference Groups of six male 0, 5, 10, 20 mg/kg bw No significant diuretic Varner et al. Sprague-Dawley rats per day orally in activity (1990a) distilled water Groups of two male 5 mg/kg bw per day Mild transient decreases Gromelski et al. and two female intravenously in blood pressure, cardiac (1990) beagle dogs output, left ventricular pressure, and left ventricular end diastolic pressure in two dogs. No effects on electro- cardiographic wave forms Groups of three male 1, 10, 100, 1000 mg/kg No effect on central or Varner et al. Sprague-Dawley rats bw per day orally peripheral nervous system (1990b) at 100 mg/kg bw per day; salivation and tremors at 1000 mg/kg bw per day Groups of eight male 0, 5, 10, 20 mg/kg bw 18, 27, and 23% decreases Varner et al. CD-1 mice per day orally in in gastrointestinal motility in (1990c) distilled water; 4 mg/kg comparison with vehicle morphine sulfate (positive control control) Groups of eight 0, 5, 10, 20 mg/kg bw Increased gastric fluid Varner et al. pyloricligated male per day intraduodenally volume at all doses (1990d) Sprague-Dawley rats in 0.25% methylcellulosea; (not dose-related); 10 mg/kg bw per day increased gastric acidity cimetidine (positive at all doses control) a The solvent vehicle was reported to be 0.25% methylcellulose in some parts of the report and distilled water in others. 2.2.8 Special studies on microbiological effects In a study carried out according to the methods described in documents M11A (1979) and M11A2 (1990) of the National Committee for Clinical Laboratory Standards, USA, the antibacterial activity of danofloxacin and desmethyldano-floxacin was determined against 64 isolates of six genera of organisms representative of the human intestinal anaerobic microflora. In order to assess the reproducibility of the assay, four reference strains, Bacterides fragilis ATCC 25285, Bacillus thetaiomicron ATCC 29741, Clostridium perfringens ATCC 13124, and Eubacterium lentum ATCC 43055, were added to each batch of tests. In addition, data were provided for the facultative anaerobes Lactobacillus spp (14 strains) and Proteus spp (11 strains) isolated from faeces of patients in the hospital of Tourcoing, France. Escherichia coli ATCC 25922 and Enterococcus faecalis ATCC 29242 were added as reference strains. The calculated MIC50 values are shown in Tables 9 and 10. 2.3 Observations in humans No information was available. Danofloxacin is not authorized for human use. Table 9. MIC50 values for danofloxacin and desmethyldanofloxacin in vitro against some strains of bacteria representative of those found in the human gut Species No. of Inoculum MIC50 (µg/ml) strains density (cfu/ml) Danofloxacin Desmethyldanofloxacin Anaerobes Bacillus fragilis group 12 107-108 4 128 Fusobacterium spp. 10 4 16 Clostridium spp. 10 0.5 0.5 Eubacterium spp. 10 0.5 1 Bifidobacterium spp. 10 2 8 Peptostreptococcus spp. 12 0.5 2 Facultative anaerobes Lactobacillus spp. 14 106 16 > 128 Proteus spp. 11 106 0.25 0.06 Table 10. MIC50 values for danofloxacin and desmethyldanofloxacin in vitro against some strains used for quality control Species Anaerobic incubation Aerobic incubation Danofloxacin Desmethyldanofloxacin Danofloxacin Desmethyldanofloxacin Escherichia coli 0.06 0.06 0.03 0.015 ATCC 25922 Enterococcus faecalis 1 4 2 1 ATCC 29242a a Shown as Enterococcus faecalis ATCC 29242 on p. 5 of the report but as Enterococcus faecalis ATCC 29212 in the accompanying tables (Dubreuil, 1994) 3. COMMENTS The Committee considered data from studies on the pharmacokinetics, acute, short-term, and long-term toxicity, carcinogenicity, reproductive toxicity, genotoxicity, and antimicrobial activity of danofloxacin. The results of studies on the acute and short-term toxicity, reproductive toxicity, genotoxicity, and antimicrobial activity of the metabolite desmethyldanofloxacin were also reviewed. Most of the studies critical for the evaluation were carried out in accordance with appropriate standards for study protocol and conduct. Danofloxacin was rapidly absorbed after oral administration to chickens and pigs and after intramuscular administration to pigs and cattle. Only one study was carried out in which the oral bioavailability of danofloxacin was calculated; in this study, the bioavailability after oral administration of 5 mg/kg bw to pigs was approximately 90%. The substance was well distributed to the tissues. Urine and faeces contained approximately equal amounts of danofloxacin and its metabolites. In cattle, dogs, and rats, unchanged danofloxacin was the main substance present in the faeces; smaller amounts of desmethyldanofloxacin were found. Danofloxacin was also the main component in urine. Desmethyldanofloxacin, danofloxacin- N-oxide, and the ß-glucuronide were also found in urine. A piperazine-ring degradation product was present in the bile of cattle but was found in only trace amounts in the bile of rats and dogs. Residues of both danofloxacin and desmethyldanofloxacin were found in liver samples from rats, dogs, and the three target species. The Committee considered that the metabolism of danofloxacin was very similar in laboratory animals and in the three target species. Single oral doses of both danofloxacin and desmethyldanofloxacin were slightly toxic to rats and mice (acute oral LD50 values in the range 1500 to > 2000 mg/kg bw). Signs of toxicity typical of stimulation of the central nervous system were observed prior to death. Several exploratory studies were carried out in rats given danofloxacin in the feed and by gavage and in rabbits given danofloxacin by gavage. Caecal dilatation was seen at doses of 25 mg/kg bw per day and above in most of these studies. In rats, increased incidences of crystals in the urine were observed at doses of 75 mg/kg bw per day and above. There were no corresponding pathological changes in the kidneys. In a three-month study in which danofloxacin was administered to rats in the diet, degenerated germinal cells were found in the epididymides of all males given 300 mg/kg bw per day and in 9 out of 15 males given 150 mg/kg bw per day. In view of the high doses used, the results of these studies were not useful in evaluating the safety of danofloxacin. Rats were exposed to danofloxacin in utero and during lactation by administration to the dams of doses of 0, 25, 75, or 150 mg/kg bw per day. The rats were orally dosed at the same levels for an additional three months after weaning. In females, there was a dose-related increase in proteinuria which correlated with the finding of tubular nephropathy in individual animals. In males at 75 and 150 mg/kg bw per day, both the mean absolute and relative testicular weights were 10% lower than those of controls. In a follow-up three-month study with lower doses, the highest dose, 6.25 mg/kg bw per day, was the NOEL, on the basis of renal tubular nephropathy. Rats were exposed to desmethyldanofloxacin in utero and during lactation by administration to the dams of oral doses of 0, 1, 2.5, or 6.25 mg/kg bw per day. The rats were treated orally at the same levels for an additional three months after weaning. No adverse effects were observed at any dose. Six-month-old dogs were given oral doses of 0, 5, 10, or 25 mg/kg bw per day of danofloxacin in gelatin capsules for three months. Those given the two higher doses showed signs of joint pain. Pathological examination revealed arthropathy characterized by areas of cartilage separation and erosion in all treated groups, and the severity of the lesions was dose-related. A second three-month study was carried out in immature dogs given oral doses of 0, 1, or 2.4 mg/kg bw per day danofloxacin in gelatin capsules. There was no evidence of arthropathy or any other treatment-related effect. On the basis of the results of the two studies, the Committee concluded that 2.4 mg/kg bw per day was the overall NOEL for arthropathy in dogs. A further three-month study was carried out in which immature dogs were given oral doses of 0, 2.5, 5, or 10 mg/kg bw per day desmethyldanofloxacin in gelatin capsules. One male of three given the highest dose and one female of three given the lowest dose showed signs of pain on examination. In the male, morphological changes were found in the articular cartilage of one joint. When the study was repeated at doses of 0, 0.25, or 0.5 mg/kg bw per day desmethyldanofloxacin, one male of three given 0.5 mg/kg bw per day was found to have histopathological changes in the right knee typical of quinolone-induced arthropathy. The NOEL for desmethyldanofloxacin was 0.25 mg/kg bw per day, on the basis of arthropathy. In a 102-day range-finding study, mice were fed diets containing danofloxacin at concentrations equivalent to 0, 150, 300, or 600 mg/kg bw per day. At the highest dose, the body-weight gain of males was reduced, and decreased haematological parameters and increased kidney weights were observed in females. Caecal dilatation was observed at all doses, and two of 30 mice given the highest dose had inflammation of the caecum. In a two-year study of carcinogenicity, mice were fed diets containing doses equal to 0, 10, 50, or 100 mg/kg bw per day. Females at the highest dose gained more weight than the controls and showed increased absolute kidney weights. There were no adverse effects on haematological parameters, but clinical chemistry was not monitored. No increase in tumour incidence was observed at any dose. In a two-year study of carcinogenicity, rats were fed diets providing doses of 0, 10, 50, or 100 mg/kg bw per day danofloxacin. At the highest dose, mean haemoglobin, haematocrit, and lymphocyte counts were decreased in females, and serum aspartate aminotransferase activity was increased and serum globulin levels were reduced in males. Serum sorbitol dehydrogenase activity was increased in males given 50 or 100 mg/kg bw per day. The relative testicular weight was reduced in rats at the highest dose, and increased oligospermia and abnormal epididymal content were observed in this group. Caecal enlargement was noted in all treated groups but was not correlated with any microscopic findings. An increased incidence of papillary oedema was seen in the kidneys of rats at 100 mg/kg bw per day. There was a significant positive trend in the incidence of granular-cell tumours of the uterus and vagina in treated female rats. Tumours were distinguished from foci by their larger size and the compression of adjacent tissue, but the morphology of these lesions was similar, and the Committee considered that it was appropriate to combine the incidences of these uterine and vaginal foci and tumours. There was no significant trend in the combined incidence across groups. There was a significant positive trend in the incidence of pituitary adenomas in females, but the number of rats with these lesions was within the range in historical controls. In addition, none of the trends in tumour incidence was significant when corrected for multiple comparisons. The Committee concluded that neither the granular-cell lesions of the uterus and vagina nor the pituitary adenomas were indicative of a carcinogenic response to treatment with danofloxacin. The genotoxic properties of danofloxacin were investigated in vitro in assays for gene mutation in bacteria and in mammalian cells, unscheduled DNA synthesis, and cytogenetics alterations in mammalian cells and for cytogenetic effects in vivo. All of the tests gave negative results, except for an assay of cytogenetic alterations in human lymphocytes in vitro. The clastogenicity observed in vitro was reduced or abolished by addition of magnesium sulfate to the culture medium and/or washing the cells after treatment to remove danofloxacin, and appeared to be associated with the cation- chelating properties of danofloxacin. There was no evidence of clastogenicity in vivo. Desmethyldanofloxacin induced a significant increase in unscheduled DNA synthesis in two independent assays in primary rat hepatocytes in vitro; however, negative results were obtained with desmethyldanofloxacin in an assay for unscheduled DNA synthesis and in a test for micronucleus formation in vivo. Thus, although desmethyldanofloxacin induced unscheduled DNA synthesis, this genotoxic potential did not appear to be expressed in vivo. In a two-generation study of reproductive toxicity, rats received doses of 0, 25, 75, or 150 mg/kg bw per day of danofloxacin by gavage. In the parental generation, maternal body-weight gain was reduced at the highest dose and these dams had fewer implantation sites and produced fewer viable pups. The effects were observed at lower doses with subsequent matings. At the second mating of the first-generation animals, the pregnancy rate was adversely affected in all treated groups. No NOEL could be identified. A three-generation study of reproductive toxicity was carried out in which rats were given danofloxacin at doses of 0, 1, 2.5, 6.25, or 150 mg/kg bw per day by gavage. At 150 mg/kg bw per day, the number of mated females and the pregnancy rate were reduced, the duration of gestation was increased and the litter sizes and pup weights were reduced; this group was terminated before the second mating of the first generation. In this study, in which lower doses were used than in the study described above, the NOEL for reproductive toxicity was 6.25 mg/kg bw per day. No adverse effects were observed in a three-generation study of reproductive toxicity in rats with desmethyldanofloxacin in which doses of up to 6.25 mg/kg bw per day were administered by gavage. In a study of developmental toxicity in mice, there was no evidence of teratogenicity when danofloxacin was administered by gavage at doses of up to 200 mg/kg bw per day on days 6-13 of gestation. The dose of 200 mg/kg bw per day was toxic to the dams, reducing body-weight gain, and was fetotoxic, producing a reduction in mean fetal weight and an increased incidence of delayed ossification. The study was compromised by the small numbers of gravid dams in all groups, including the controls. The NOEL for both maternal and fetal toxicity was 100 mg/kg bw per day. Oral doses of 0, 50, 100, or 200 mg/kg bw per day were administered to rats on days 6-15 of gestation in a study of developmental toxicity. Maternal body-weight gain and food consumption were reduced in animals at 100 or 200 mg/kg bw per day. At these doses, the incidences of delayed ossification and dilatation of the cerebral ventricles were significantly increased. The NOEL for both maternal and fetal toxicity was 50 mg/kg bw per day. In a study of developmental toxicity, oral doses of 0, 2.5, 7.5, or 15 mg/kg bw per day danofloxacin were administered to rabbits on days 6-20 of gestation. At a dose of 15 mg/kg bw per day, maternal body-weight loss, reduced food consumption, and abortion were observed. The NOEL for maternal toxicity was 7.5 mg/kg bw per day. The minimum concentration of danofloxacin resulting in 50% inhibition (MIC50) was determined for 64 isolates of the six predominant genera of human intestinal anaerobic microflora ( Bacteroides, Fusobacterium, Clostridium, Eubacterium, Bifidobacterium, and Peptostreptococcus). In addition, data were provided for the facultative anaerobes Lactobacillus, Proteus, and Escherichia coli. Although E. coli and Proteus were the most sensitive organisms, the Committee agreed that they should not be taken into account in the calculation of the MIC50 because they are not predominant species in the human intestine. Instead, the Committee derived the mean MIC50 from the data available on 32 strains of the most sensitive relevant genera isolated from the human gastrointestinal tract, in this case Eubacterium spp., Bifidobacterium spp., and Peptostreptococcus spp. The mean MIC50 for these strains was 1 µg/ml. This figure was used in calculating the upper limit of the ADI from the formula described on p. 12: Upper limit 1 µg/ga × 220 g = of ADI 0.1b × 1c × 60 kg = 37 µg/kg bw a Mean MIC50 for the most sensitive relevant species in the human intestine, in this case Eubacterium spp., Bifidobacterium spp., and Peptostreptococcus spp. b Bioavailability was determined to be about 90%, based on a study in pigs given an oral dose of 5 mg/kg bw. The finding that danofloxacin is strongly bound to cattle faeces was considered by the Committee to add confidence to use of this value. c A safety factor of 1 was used because sufficient relevant microbiological data were provided. The metabolite desmethyldanofloxacin was one-quarter to one-half as active as danofloxacin against the same isolates. 4. EVALUATION The Committee noted that danofloxacin belongs to a group of fluoroquinolones that is active against aerobic gram-negative bacteria and that the main components of the flora in the human gastrointestinal tract are largely unaffected by these compounds. Therefore, the Committee decided to base the ADI on the toxicity of danofloxacin and not on its effects on the intestinal flora. Moreover, the toxicological end-point resulted in a lower ADI. The Committee established an ADI of 0-20 µg/kg bw per day on the basis of the NOEL of 2.4 mg/kg bw per day for arthropathy in a three-month study in immature dogs and a safety factor of 100. The ADI was rounded to one significant figure, as is the standard practice (Annex 1, reference 91, section 2.7). The Committee noted that the NOEL for arthropathy in studies with desmethyldanofloxacin was 0.25 mg/kg bw per day. The studies of pharmacokinetics and metabolism showed that dogs that received oral doses of danofloxacin were also exposed systemically to the major metabolite desmethyldanofloxacin. Therefore the Committee concluded that it was not necessary to calculate a separate ADI for desmethyldanofloxacin. The Committee agreed, however, that the approximately 10-fold higher toxicity of the metabolite should be taken into account when recommending MRLs, as consumers may be directly exposed to desmethyldano-floxacin in liver. 5. REFERENCES Amacher, D.E., Turner, G.N., Ellis, J.H., Holden, H.E. & Kluwe, W.M. (1988) Danofloxacin. Genetic toxicology report. Microbial reverse mutation assays. Mammalian cell gene mutation assays. Unscheduled DNA synthesis assays. Unpublished studies Nos. 86-607-01 and 87-607-02 from Pfizer Central Research, Groton, CT, USA. Submitted to WHO by Pfizer Inc., Groton, CT, USA. Amacher, D.E., Holden, H.E., Muehlbauer, P.A., Wahrenburg M.G. & Kluwe, W.M. (1990) Danofloxacin. Genetic toxicology report. Addendum. In vitro cytogenetics assays. In vivo cytogenetics assays. Unpublished study No. 89-607-18 from Pfizer Central Research, Groton, CT, USA. Submitted to WHO by Pfizer Inc., Groton, CT, USA. Amacher, D.E., Ellis, J.H. & Kluwe, W.M. (1991a) Desmethyldanofloxacin. Genetic toxicology report. Microbial reverse mutation assays. Unpublished study No. 90-576-09 from Pfizer Central Research, Groton, CT, USA. Submitted to WHO by Pfizer Inc., Groton, CT, USA. Amacher, D.E., Holden, H. E., Muehlbauer, P.A. & Kluwe, W.M. (1991b) Genetic toxicology report. Unscheduled DNA synthesis assay. Unpublished study No. 90-576-12 from Pfizer Central Research, Groton, CT, USA. Submitted to WHO by Pfizer Inc., Groton, CT, USA. Amacher, D.E., Holden, H.E., Zelljadt, I. & Kluwe, W.M. (1991c) Desmethyldano-floxacin. Genetic toxicology report. In vivo micronucleus assay. Unpublished study No. 90-576-10 from Pfizer Central Research, Groton, CT, USA. 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(1988b) Danofloxacin. One month oral capsule study in beagle dogs. Unpublished study No. 87-607-07 from Pfizer Central Research, Groton, CT, USA. Submitted to WHO by Pfizer Inc., Groton, CT, USA. Fisher, D.O., Santacroce, C.E., Mayne, J.T., Milisen, W.B., Estes, P.C. & Levinsky, H.V. (1989a) Danofloxacin. Three months oral gavage study with in utero exposure in Long-Evans rats. Unpublished study No. 88-607-12 from Pfizer Central Research, Groton, CT, USA. Submitted to WHO by Pfizer Inc., Groton, CT, USA. Fisher, D.O., Soler, G.N., Mayne, J.T., Walsh, A.H., Levinsky, H.V. & Estes, P.C. (1989b) Danofloxacin. Three month oral capsule study in beagle dogs. Unpublished study No. 88-607-14 from Pfizer Central Research, Groton, CT, USA. Submitted to WHO by Pfizer Inc., Groton, CT, USA. Fisher, D.O., Santacroce, C.E., Carson, D.L., Son, D.L., Walsh, A.H., Levinsky, H.V. & Estes, P.C (1989c) Danofloxacin. Three month oral capsule study in beagle dogs. 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Fetotoxicity study in mice by the oral route. Segment II. Unpublished studies No. 88115 and 88116 from Pfizer Central Research, Amboise Cedex, France. Submitted to WHO by Pfizer Inc., Groton, CT, USA. Kesseddjian, M.J., Stadler, J. & Paulus, G. (1989b) Danofloxacin. Fetotoxicity study in rats by the oral route. Segment II. Unpublished studies No. 88093 and 88094 from Pfizer Central Research, Amboise Cedex, France. Submitted to WHO by Pfizer Inc., Groton, CT, USA. Lundeen, G.R., Macaione, G.M., Santacroce, E.M. & Rice, J.R. (1993) Two week exploratory toleration study in New Zealand white rabbits. Unpublished study No. 92-607-30 from Pfizer Central Research, Terre Haute, IN, and Groton, CT, USA. Submitted to WHO by Pfizer Inc., USA. Lynch, M.J., Ronfield, R.A., Rainier, R.H., Magonigle, R.A., Frame, G.M., Risk, J.E., Mosher, F.R. & Harran, L.P. (1988) Danofloxacin. Nonclinical total residue depletion study in broiler chickens. Unpublished study No. 1515N-60-88-006 from Pfizer Central Research, Terre Haute, IN, and Groton, CT, USA. Submitted to WHO by Pfizer Inc., Groton, CT, USA. Lynch, M.J., Ronfield, R.A., Rainier, R.H., Magonigle, R.A., Frame, G.M., Risk, J.E., Mosher, F.R. & Harran, L.P. (1989a) Danofloxacin. Nonclinical total residue excretion study in cattle. Unpublished study No. 1535N-60-88-008 from Pfizer Central Research, Terre Haute, IN, and Groton, CT, USA. Submitted to WHO by Pfizer Inc., Groton, CT, USA. Lynch, M.J., Ronfield, R.A., Rainier, R.H., Magonigle, R.A., Frame, G.M., Risk, J.E., Mosher, F.R. & Harran, L.P. (1989b) Danofloxacin. A nonclinical total residue excretion study in swine. Unpublished study No. 1525C-60-88-009 from Pfizer Central Research, Terre Haute, IN, and Groton, CT, CT. Submitted to WHO by Pfizer Inc., Groton, CT, USA. Lynch, M.J., Ronfield, R.A., Rainier, R.H., Magonigle, R.A., Frame, G.M., Risk, J.E., Mosher, F.R. & Harran, L.P. (1989c) Danofloxacin. A nonclinical total residue depletion study in swine. Unpublished study No. 1525N-60-88-008 from Pfizer Central Research, Terre Haute, IN, and Groton, CT. Submitted to WHO by Pfizer Inc., Groton, CT, USA. Lynch, M.J., Ronfield, R.A., Magonigle, R.A., Risk, J.E., Rice, J.R., Millas, W.J. & Harran, L.P. (1990a) Danofloxacin. Plasma and lung pharmacokinetics study in broiler chickens. Unpublished Study No. 1514C-60-90-002 from Pfizer Central Research, Terre Haute, IN, and Groton, CT, USA. Submitted to WHO by Pfizer Inc., Groton, CT, USA. Lynch, M.J., Ronfield, R.A., Magonigle, R.A., Risk, J.E., Rice, J.R., Horan, M.R. & Gummerus, J.A. (1990b) Danofloxacin. Nonclinical plasma and lung pharmacokinetics study in cattle. Unpublished study No. 1534N-60-89-003 from Pfizer Central Research, Terre Haute, IN, and Groton, CT, USA. Submitted to WHO by Pfizer Inc., Groton, CT, USA. Lynch, M.J., Ronfield, R.A., Magonigle, R.A., Risk, J.E., Rice, J.R. & Harran, L.P. (1991a) Danofloxacin. Determination of plasma pharmacokinetics and bioavailability in cattle. Unpublished study No. 1532N-60-89-005 from Pfizer Central Research, Terre Haute, IN, and Groton, CT, USA. Submitted to WHO by Pfizer Inc., Groton, CT, USA. Lynch, M.J., Ronfield, R.A., Rainier, R.H., Magonigle, R.A., Frame, G.M., Risk, J.E., Mosher, F.R. & Harran, L.P. (1991b) Danofloxacin. Nonclinical total residue excretion study in broiler chickens. Unpublished study No. 1515N-60-88-007 from Pfizer Central Research, Terre Haute, IN, and Groton, CT, USA. Submitted to WHO by Pfizer Inc., USA. Lynch, M.J., Ronfield, R.A., Magonigle, R.A., Risk, J.E., Rice, J.R., Millas, W.J. & Harran, L.P. (1991c) A plasma and lung pharmacokinetics study in swine. Unpublished study No. 1524C-60-89-003 from Pfizer Central Research, Terre Haute, IN, and Groton, CT, USA. Submitted to WHO by Pfizer Inc., Groton, CT, USA. Lynch, M.J., Ericson, J., Calcagni, A., Gummerus, J. & Nowakowski, M.A. (1991d) The comparative metabolism of 3H-danofloxacin in cattle, dog and rat species. Unpublished study No. CM-90-02 from Pfizer Central Research, Terre Haute, IN, and Groton, CT, USA. Submitted to WHO by Pfizer Inc., Groton, CT, USA. Mann, D.D. & Frame, G.M (1992) Pharmacokinetic study of danofloxacin in cattle and swine. Am. J. Vet. Res., 53, 1022-1026. Reynolds, J.A., Saunders, W., Hatch, G.W. & Vessella, D.J. (1987a) 2 week oral gavage toleration study in Long-Evans rats. Unpublished study No. 87-607-03 from Pfizer Central Research, Terre Haute, IN, and Groton, CT. Submitted to WHO by Pfizer Inc., Groton, CT, USA. Reynolds, J.A., Milisen, W.B. & Frame, G.M. (1987b) Two week oral capsule toleration study in beagle dogs. Unpublished study No. 87-607-05 from Pfizer Central Research, Terre Haute, IN, and Groton, CT. Submitted to WHO by Pfizer Inc., Groton, CT, USA. 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Acute oral and intravenous toxicity studies in albino mice and Sprague-Dawley rats. Unpublished study No. 89-576-05 from Pfizer Central Research, Groton, CT, USA. Submitted to WHO by Pfizer Inc., Groton, CT, USA. Stadnicki, S.W., Cacciatore, B.A., Lundeen, G.R., Rice, J., Roesler, A.R., Estes, P.C. & Levinsky, H.V. (1990a) Desmethyldanofloxacin. Three month gavage study in Long-Evans rats with in utero exposure. Unpublished study No. 89-576-06 from Pfizer Central Research, Groton, CT, USA. Submitted to WHO by Pfizer Inc., Groton, CT, USA. Stadnicki, S.W., Engel, S.M., Blackwell, D.K., Rice, J.R., Walsh, A.H., Estes, P.C. & Levinsky, H.V. (1990b) Desmethyldanofloxacin. Three months oral capsule study in beagle dogs. Unpublished study No. 89-576-03 from Pfizer Central Research, Groton, CT, USA. Submitted to WHO by Pfizer Inc., Groton, CT, USA. Stadnicki, S.W., Engel, S.M., Blackwell, D.K., Rice, J.R., Estes, P.C. & Levinsky, H.V. (1990c) Desmethyldanofloxacin. 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Unpublished study No. 92-607-29 from Pfizer Central Research, Groton, CT, USA. Submitted to WHO by Pfizer Inc., Groton, CT, USA. Stadnicki, S.W., Cacciatore, A., Rovetti, C.C., Davenport, C.J., Chatman, L.A. & Rice, J.R. (1994b) One month feeding study in Fischer rats. Unpublished study No. 91-607-23 from Pfizer Pfizer Central Research, Groton, CT, USA. Submitted to WHO by Pfizer Inc., Groton, CT, USA. Stadnicki, S.W., Cacciatore, A., Davenport, C.J., Coleman, G.L. & Rice, J.R (1994c) Three week feeding study in Long-Evans rats. Unpublished study no. 92-607-25 from Pfizer Central Research, Groton, CT, USA. Submitted to WHO by Pfizer Inc., Groton, CT, USA. Stadnicki, S.W., Boldt, B.K., Rovetti, C.C., Davenport, C.J., Milisen, W.B. & Rice, J.R. (1994d) Three month feeding study in Long-Evans rats. Unpublished study No. 92-607-28 from Pfizer Central Research, Groton, CT, USA. Submitted to WHO by Pfizer Inc., Groton, CT, USA. 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See Also: Toxicological Abbreviations DANOFLOXACIN (JECFA Evaluation)